**3.2 Bioethanol production by** *Saccharomyces cerevisiae*

#### **3.2.1 Materials and methods**

#### **3.2.1.1 Microorganisms**

The yeast *Saccharomyces cerevisiae* B-4 obtained from Institute of Agricultural and Food Biotechnology Warsaw, Poland, was used for assessment ultrasound exposition to ethanol production. The yeast cultures were cultivated on YPG slants (2% glucose, 2% peptone, 1% yeast extract) supplemented with 2% agar, at pH 5.0 and 30 ºC for 24 h. The active cultures

Feasibility of Bioenergy Production from

frequency of 20 kHz.

another.

**3.2.1.3 Analytical methods** 

**3.2.2 Results and discussion 3.2.2.1 Sonification parameters** 

each fermentation step and the aged one was removed.

triplicates and results were reproducible within 3% deviation.

Ultrafiltration Whey Permeate Using the UASB Reactors 205

when the standard deviations of the ethanol and lactose concentrations in the effluent distillate were within 3%), thus 30 days of each fermentation step (step 1 – HRT of 12 h, step 2 – HRT of 24 h, step 3 – HRT of 36 h). The fresh inoculum was added to the reactors before

The ultrasound irradiation of the reactor with yeasts was made by a special ring with a transducer (Intersonic S.C. Poland) that was attached at the bottom of the reactor. The range of the frequency generator was adjustable between 20–25 kHz and the maximum power of 50 W. The experiments were carried out with the stable sonication power of 1 W L-1 and the

Lactose and ethanol concentrations in the effluent distillate were determined according to Standard Methods (PN-67/A-86430; PN-A-79528-3:2007). The samples were analyzed in

All fermentation steps connected with different HRTs were carried out in triplicate. Significant differences between the effects obtained in the two reactors with and without ultrasound exposure were analysed using an ANOVA *F*-test (Statistica 7.1 software, Statsoft Inc.) A 5% probability level was applied for all the tests. If p<0.05 from an ANOVA *F*-test, the differences between the effects were considered to be significantly different from one

In the experiment, the frequency of applied ultrasounds was 20 kHz and the power input was 1.0 W L-1. The initial experiments were done to find the best irradiation period. The experiments revealed, that continuous low energy ultrasound irradiation during 12, 24 and 36 h did not enhance ethanol productivity by co-immobilized *S. cerevisiae*, moreover the ethanol yield coefficients were lower than those obtained in experiments without ultrasound irradiation. The subsequent experiments were carried out with time intervals with and without ultrasonic irradiation in order to obtain the positive influence of ultrasound on biological activity of *S. cerevisiae*. The results showed that the culture should have been sonicated for 1 min every 6 h. It was similarly to results obtained by Marques et al. (2006). They investigated the effect of ultrasound pulses on enzymatic activity of *S. cerevisiae*. Their results showed that the ultrasound pulse at low frequency (20–25 kHz) for a short sonification period of 1 and 2 min increased cell permeability, and the viability rate of yeasts

The use of ultrasounds to stimulate biological activity and ethanol production by *S. cerevisiae*  are reported by Schläfer et al. (2000). After testing several different frequencies and power levels, they carried out the experiments at 25 kHz, 0.3 and 12 W L-1. At an ultrasound intensity of 12 W L-1 there was no recognizable difference in the biological activity of yeasts with and without ultrasound. The authors stated that some pauses are needed between ultrasound exposure to obtain positive effects on biological activity of yeast *S. cerevisiae*. Moreover, an increase in biological activity appeared after irradiation and high activity of ultrasound activated cultures stopped for some hours after irradiation. The authors stated,

was over 95%. However, in the 4 min sonification, the rate decreased to 46%.

for inoculation were prepared by growing the yeast in a 1 L baffled shake-flask containing sterile water and 100 mL YPG medium at 30 ºC for 24 h on orbital shaker table at 200 rpm to a concentration of approximately 108 cells mL-1. The cultures in baffled shaken flasks of 100 mL were used to prepare the inocula. After 24 h of incubation at 30 ºC, the precultures were centrifuged at 3800 rpm for 10 min and the cells were resuspended in sterile water to obtain 106 cells mL-1. Enzyme β-D-galactosidase (optimum temperature 30 ºC, optimum acidity pH 4.5-5.0, activity 8.7 AU mg-1 d.m. of the preparation), from *Aspergillus oryzae* manufactured by the SIGMA company (USA), was used for co-immobilization process. The amount of yeast and enzyme was 3% free cell inoculum and 4 cm3 enzyme solution. The yeast culture was co-immobilized in the 2% (w/v) sodium alginate by dropping yeast and enzyme into 150 cm3 0.09 mol L-1 solution of CaCl2 with 10% glucose. The cell beads were washed with sterile water and were stored as a fermentation medium in physiological solution at 8C.

#### **3.2.1.2 Fermentation medium and experimental system**

UF whey permeate (non-deproteinized, non diluted and non-sterilized) with the average lactose concentration of 50 g L-1 from the Dairy Plant in Nowy Dwór Gdański, Poland, was used as a fermentation substrate (Table 2).

Continuous fermentation was carried out in the laboratory-scale plant consisted of the two UASB reactors with a working volume of 5 L each (Fig. 8). These two reactors were used to enable parallel test series with and without ultrasound irradiation. The fermentation medium was pumped continuously to the bottom part of the reaction tank by means of the peristaltic pumps. The necessary mixing was achieved through the upward wastewater flow. The reactors were water-jacketed and operated at a constant temperature of 30±1 ºC. The pH of mixed liquid in the reactors was controlled automatically at pH 5.1 ± 0.2 with 2 M NaOH.

Fig. 8. A scheme of the research station.

The reactors were inoculated with 40% (v/v) solid beads containing the immobilized cells which corresponded to 39.4 g cells dry weight - DW L-1 of working bioreactor volume. After adding the cell beads inoculum to the bioreactors, before starting continuous feeding, a batch fermentation was conducted for 24 h under additional gentle agitation (100 rpm). Next the reactors worked at different HRTs of 12, 24 and 36 h. At each HRT the reactor was operated till it has reached the steady-state (the steady-state conditions were evidenced when the standard deviations of the ethanol and lactose concentrations in the effluent distillate were within 3%), thus 30 days of each fermentation step (step 1 – HRT of 12 h, step 2 – HRT of 24 h, step 3 – HRT of 36 h). The fresh inoculum was added to the reactors before each fermentation step and the aged one was removed.

The ultrasound irradiation of the reactor with yeasts was made by a special ring with a transducer (Intersonic S.C. Poland) that was attached at the bottom of the reactor. The range of the frequency generator was adjustable between 20–25 kHz and the maximum power of 50 W. The experiments were carried out with the stable sonication power of 1 W L-1 and the frequency of 20 kHz.
